Inhibition Of Insulin Biosynthesis Research Articles

Prednisolone-induced beta cell dysfunction is associated with impaired endoplasmic reticulum homeostasis in INS-1E cells

Glucocorticoids (GCs), such as prednisolone (PRED), are widely prescribed anti-inflammatory drugs, but their use may induce glucose intolerance and diabetes. GC-induced beta cell dysfunction contributes to these diabetogenic effects through mechanisms that remain to be elucidated. In this study, we hypothesized that activation of the unfolded protein response (UPR) following endoplasmic reticulum (ER) stress could be one of the underlying mechanisms involved in GC-induced beta cell dysfunction. We report here that PRED did not affect basal insulin release but time-dependently inhibited glucose-stimulated insulin secretion in INS-1E cells. PRED treatment also decreased both PDX1 and insulin expression, leading to a marked reduction in cellular insulin content. These PRED-induced detrimental effects were found to be prevented by prior treatment with the glucocorticoid receptor (GR) antagonist RU486 and associated with activation of two of the three branches of the UPR. Indeed, PRED induced a GR-mediated activation of both ATF6 and IRE1/XBP1 pathways but was found to reduce the phosphorylation of PERK and its downstream substrate eIF2α. These modulations of ER stress pathways were accompanied by upregulation of calpain 10 and increased cleaved caspase 3, indicating that long term exposure to PRED ultimately promotes apoptosis. Taken together, our data suggest that the inhibition of insulin biosynthesis by PRED in the insulin-secreting INS-1E cells results, at least in part, from a GR-mediated impairment in ER homeostasis which may lead to apoptotic cell death.

Syntaxin-3 and syntaxin-1A inhibit L-type calcium channel activity, insulin biosynthesis and exocytosis in beta-cell lines.

Syntaxin-1A (Syn-1A) is known to play a negative regulatory role in insulin secretion but the precise mechanisms for its action are not clear. Syn-2, -3 and -4 are also present in islet beta cells but their functions are not known. Here, we investigated the role of these syntaxins in the insulin secretory process. We examined the following effects of Syn-1, -2, -3 and -4 expression in insulinoma beta-cell lines. Endogenous insulin secretion was measured by batch radioimmunoassay (RIA) and single cell patch clamp capacitance measurements. The L-type Ca(2+) channel activity was studied by patch clamp electrophysiology. Insulin gene transcription was examined by Northern blotting and measurement of insulin gene promoter activity by the co-expression of cyan fluorescent protein-labelled rat insulin promoter. Syn-1A or -3, but not Syn-2 or -4 overexpression, inhibited K(+)-induced insulin release as determined by RIA (49.7 +/- 5.5 % and 49.1 +/- 6.2 %, respectively) and electrophysiologic membrane capacitance measurements (68.0 +/- 21.0 % and 58.0 +/- 13.2 %, respectively). Overexpressed Syn-1A and -3, but not Syn-2, inhibited Ca(2+) channel current amplitude by 39.5 +/- 11.6 % and 52.7 +/- 6.0 %, respectively. Of note, overexpression of Syn-1A and -3 also reduced single cell (by confocal microscopy) and total cellular endogenous insulin content (by RIA) by 24.8 +/- 4.2 % and 31.8 +/- 3.9 %, respectively. This correlated to a reduction in endogenous insulin mRNA by 24.5 +/- 4.2 % and 25.7 +/- 4.2 %, respectively. This inhibition of insulin biosynthesis is mainly at the level of insulin gene transcription as demonstrated by an inhibition of insulin gene promoter activity (53.3 +/- 9.15 % and 39.0 +/- 6.8 %, respectively). These results demonstrate that Syn-1A and -3 possess strong inhibitory actions on both insulin exocytosis and insulin biosynthesis whereas Syn-2 and -4 do not inhibit the insulin secretory process.

Pancreatic and nephrotoxicity of immunomodulator compounds

Increasing clinical experience of FK 506 in transplantation therapy has revealed a number of potentially restrictive adverse effects associated with its use. The mechanisms of action underlying 2 prominent toxic effects of FK 506, namely diabetogenesis and renal dysfunction, were investigated. A simple model system based on the effect of FK 506 on isolated rat pancreatic islets was utilised to study the relationship between inhibition of insulin biosynthesis, inhibition of interleukin 2 (IL-2) activation and FK binding protein (FKBP-12) binding of FK 506 and a number of FK 506 analogues. Results indicate that the action of these compounds on inhibition of insulin biosynthesis (and by implication, diabetogenesis) may be related to their immunosuppressive potential. Observations on the FK 506-induced release of endothelin-1 from isolated rat kidney mesangial cells suggest that this cell may be an important target associated with the nephrotoxic potential of the drug, and that this action may be mediated via the FKBP.

Inhibition of insulin production by cyproheptadine in RINm5F rat insulinoma cells.

The clonal insulin producing cell line RINm5F was evaluated as a model for the action of cyproheptadine (CPH)-like diabetogenic compounds in the rat pancreas. Treatment with 10 microM CPH and selected structural analogs under culture conditions produced a progressive loss of cellular insulin which reached 30% of control within 24 hours. Comparison of the activities of the analogs 4-diphenylmethylpiperidine (4-DPMP) and 2-diphenylmethylpiperidine (2-DPMP) to produce cellular insulin depletion showed that 4-DPMP was as active as CPH but 2-DPMP had no activity at the highest concentration employed (10 microM). The CPH metabolite desmethyl CPH-epoxide was five times more active than the parent compound in producing loss of insulin in RINm5F cells. These results are consistent with previously published results of CPH actions in vivo. An inhibition of insulin biosynthesis with no loss of preproinsulin mRNA occurred in RINm5F cells treated with CPH or DMCPH-epoxide. This suggests that an effect on transcription may not be the primary action by which CPH and its analogs inhibit insulin synthesis in vivo.

Long-term effects of leucine and arginine on B-cell function of cultivated pancreatic rat islets.

Leucine and arginine are well-known stimulators of insulin release, but they exert different actions on insulin biosynthesis in short-term incubations of isolated islets. In order to study their long-term effects of the B-cell, isolated rat islets were cultivated for 40 h in medium containing 0.5 or 3 mg glucose/ml supplemented with L-leucine (10 mmol/l) or L-arginine (10 mmol/l). After the culture the islets were incubated for 3 h with 2 mg glucose/ml without addition of the respective amino acid. Insulin biosynthesis was estimated from incorporation of [3H]phenylalanine or [3H]leucine into the (pro)insulin fraction of the islet proteins during this incubation. At the low concentrations of glucose, the leucine-cultivate islets released much more insulin than the control islets during cultures as well as during subsequent incubation. At both glucose concentrations cultivated together with this amino acid resulted in an enhanced insulin biosynthesis. The insulin-releasing effect of arginine with 0.5 mg glucose/ml was not as marked as that observed with an equimolar concentration of leucine. Islets cultivated with arginine showed no response to glucose during the incubation after culture. Hormone synthesis was found to be inhibited. Shortening of culture time to 20 h, followed by an additional 4 h cultivation without arginine did not result in an improvement of B-cell function after culture. It is concluded that leucine, in contrast with arginine, supports B-cell function, especially insulin biosynthesis, during long-term culture of islets. Stimulation of release of insulin with a concomitant inhibition of insulin biosynthesis caused by arginine during culture did not lead to a compensatory increase in hormone synthesis after culture.

Long-term exposure of isolated pancreatic islets to mannoheptulose: Evidence for insulin degradation in the β cell

The possibility that the β cell may degrade insulin to regulate pancreatic insulin stores was studied. Isolated rat islets were maintained in tissue culture for 6 days with 8.3 mM glucose and mannoheptulose (0.5, 5 and 20 mM), an inhibitor of glucose phosphorylation. Mannoheptulose inhibited glucose utilization, insulin accumulation in the culture medium, and insulin biosynthesis, and decreased islet insulin content. The total recoverable insulin from the culture system (islets plus culture medium, corrected for extracellular insulin degradation) was 251, 127, 56 and 24 per cent of the content of freshly isolated islets for 0, 0.5, 5 and 20 mM mannoheptulose, respectively. Thus both 5 and 20 mM mannoheptulose caused a net loss of insulin accounted for by intracellular degradation. After 6 days in culture with 20 mM mannoheptulose, apart from β cell degranulation, there were no ultrastructural changes indicative of the mechanism of intracellular insulin destruction. When the inhibition of insulin biosynthesis was taken into account, intracellular insulin degradation could even be demonstrated in the presence of 0.5 mM mannoheptulose. Since such degradation occurred under conditions allowing for a net increase in recoverable insulin, this may be a mechanism normally operative in islets to control insulin stores.

Perturbation of Hormone Storage and Release Induced by Cyproheptadine in Rat Pancreatic Islets in Vitro*

The effects of cyproheptadine (CPH) added in vitro were studied in rat pancreatic islets maintained in culture medium. CPH added over 6 days resulted in either an increase (5 X 10(-7) M CPH) or a marked, but reversible decrease (5 X 10(-5 M) in insulin content of islets when related to that of controls. At both concentrations, however, total recoverable insulin from islets, cells detached from islets, and medium was decreased relative to control cultures. The increased insulin content observed after 6 days with 5 X 10(-7) M CPH may be explained by the partial inhibition of insulin release, preventing the normally occurring early drop in insulin content of control islets. The decreased total recoverable insulin in the culture system with 5 X 10(-5) M CPH (17% of the initial insulin content of the islets placed into the CPH-containing culture medium) was not acounted for by the combined effects of insulin degradation in the culture medium and inhibition of insulin biosynthesis. Together and by exclusion these data suggest increased insulin degradation within beta-cells as a result of exposure to 5 X 10(-5) M CPH. Since increased intracellular insulin degradation was not found at 5 X 10(-7) M CPH, the data suggest that only severe inhibition of insulin release (5 X 10(-5) M CPH) increases intracellular insulin degradation. CPH added in vitro irreversibly decreased islet glucagon content; the data suggest that these effects are due to alterations in the physical properties of the peripheral cell layers of isolated islets. Studies with 5 X 10(-5) M CPH on the biosynthesis of insulin immunoreactive material failed to link the appearance of flocculent material in dilated cisternae of the rough endoplasmic reticulum (observed by electron microscopy) with accumulation of an immunoreactive biosynthetic precursor for insulin.